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. 2006 Apr 14;342(3):859-66.
doi: 10.1016/j.bbrc.2006.01.187. Epub 2006 Feb 17.

Human RFP2 Gene Promoter: Unique Structure and Unusual Strength

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Free PMC article

Human RFP2 Gene Promoter: Unique Structure and Unusual Strength

Mikhail Skoblov et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.

Figures

Fig. 1
Fig. 1
Schematic representation of the RFP2/RFP2OS/KCNRG locus. Non-coding exons are shown in white, RFP2 open-reading frame is in black, KCNRG open-reading frame is in grey. RFP2 promoter area studied here is shown in checkered box.
Fig. 2
Fig. 2
(A) Results of the alignment of RFP2 EST sequences with human genome sequence. Arrows indicate 5′-ends of individual cDNA clones primed on RFP2 mRNA, numbers above arrows correspond to amount of individual EST clones. (B) Transcriptional activity of the human RFP2 promoter. Fragments of indicated sizes in the 5′-region of the RFP2 gene were cloned in the pGL-Basic and transfected in HEK293 cells. The highest activity was observed with pGLM6 construct. The results are means of at least three independent experiments per construct.
Fig. 3
Fig. 3
Computationally predicted promoter elements located within the sequence of the promoter area of the human RFP2 gene. DNA fragment corresponding to insert in pGLM6 most active in luciferase assay is highlighted by blue background. First exon of the human RFP2 gene is in the underlined letters in red color, shorter isoform of this exon is shown in bold. Bold lines depict locations of the computationally predicted promoter elements. Results of the human first exon finder are underlined in blue, results of the promoter scan—in green, and NNPP prediction algorithm results in brown. Nucleotides shown in capital red letters indicate transcription start sites predicted by NNPP. Transcription Start Sites predicted by Markov chain promoter finder and by Promoter 2.0 Prediction Server are shown by green and blue arrows, respectively. Nucleotide positions shown in bold blue letters and in bold green letters are occupied by imperfect repeats forming quadruplexes at non-coding and coding DNA strands, respectively.
Fig. 4
Fig. 4
Human RFP2 promoter nucleosome binding profile (A) and the profile of electrostatic field magnitudes induced by sequence-dependent dipoles (B) for free DNA double helix produced by RFP2 promoter nucleotide sequence.

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